Montse Pérez

A Microsatellite Genetic Map of the Turbot (Scophthalmus maximus)

A consensus microsatellite-based linkage map of the turbot (Scophthalmus maximus) was constructed from two unrelated families. The mapping panel was derived from a gynogenetic family of 96 haploid embryos and a biparental diploid family of 85 full-sib progeny with known linkage phase. A total of 242 microsatellites were mapped in 26 linkage groups, six markers remaining unlinked. The consensus map length was 1343.2 cM, with an average distance between markers of 6.5 ± 0.5 cM. Similar length of female and male maps was evidenced. However, the mean recombination at common intervals throughout the genome revealed significant differences between sexes, ∼1.6 times higher in the female than in the male. The comparison of turbot microsatellite flanking sequences against the Tetraodon nigroviridis genome revealed 55 significant matches, with a mean length of 102 bp and high sequence similarity (81–100%). The comparative mapping revealed significant syntenic regions among fish species. This study represents the first linkage map in the turbot, one of the most important flatfish in European aquaculture. This map will be suitable for QTL identification of productive traits in this species and for further evolutionary studies in fish and vertebrate species.

Polymorphic microsatellite markers for blue mussels (Mytilus spp.)

Blue mussels of the genus Mytilus (M. edulis Linnaeus 1758; M. galloprovincialis Lamarck 1819; M. trossulus Gould 1850) are widely distributed in Southern and Northern hemispheres. This ecological plasticity together with the existence of interspecific hybridization in overlapping regions (Skibinski et al. 1978) makes them an interesting model for studies of population dynamics in marine habitats. Genetic marker surveys on Mytilus spp. have shown the genetic uniqueness of each species (McDonald and Koehn 1988). Several molecular markers have been developed over the last three decades to describe genetic polymorphisms in mussels. However most of them have a number of disadvantages for use as population markers, relating to data interpretation, technical difficulties, genotype discrimination, and restricted amount of polymorphism (Ohresser et al. 1997). In order to analyze intraspecific phenomena related to both natural genetic substructuring and anthropogenic changes, it is useful to develop highly informative markers, such as microsatellites.

Validation of a tRNA-Glu-cytochrome b Key for the Molecular Identification of 12 Hake Species (Merluccius spp.) and Atlantic Cod (Gadus morhua) Using PCR-RFLPs, FINS, and BLAST

The goal of this study was to develop a diagnostic key for hake meat to solve the limitations of previous identification methodologies, mainly related to the high degradation of the DNA recovered from processed foods. We describe the development of two molecular tools based on polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphisms of the cytochrome b gene, respectively, to identify DNA from 12 hake species in commercial products. The first assay is an exclusion test consisting of the PCR amplification of a 122 bp fragment using nested primers interspecifically conserved in Merluccius spp. and in Gadus morhua. This 122 bp amplicon, being the shortest one so far designed for hake DNA, is a useful traceability tool for highly degraded samples because its sequence contains enough interspecific diagnostic variation to identify 10 hake species and cod and has been successfully amplified from most commercial products so far tested. The second identification key follows a positive outcome of the exclusion test and consists of the PCR amplification of a 464-465 bp fragment and its digestion with three restriction enzymes whose targets map at interspecifically nonconserved sites of the cytochrome b. The key presented here has passed through a rigorous methodological calibration including its testing for genus specificity, its validation on a large number of authenticated sample types from each species range, and its implementation with a maximum likelihood method for the assignment of unknown samples. Together, these two procedures constitute the most complete molecular key so far developed for Merluccius spp., which is optimal for routine identification of hakes in large commercial samples at a reasonable cost-time ratio.

Activation of TREK Currents by the Neuroprotective Agent Riluzole in Mouse Sympathetic Neurons

Background K2P channels play a key role in stabilizing the resting membrane potential, thereby modulating cell excitability in the central and peripheral somatic nervous system. Whole-cell experiments revealed a riluzole-activated current (IRIL), transported by potassium, in mouse superior cervical ganglion (mSCG) neurons. The activation of this current by riluzole, linoleic acid, membrane stretch, and internal acidification, its open rectification and insensitivity to most classic potassium channel blockers, indicated that IRIL flows through channels of the TREK [two-pore domain weak inwardly rectifying K channel (TWIK)-related K channel] subfamily. Whole-ganglia and single-cell reverse transcription-PCR demonstrated the presence of TREK-1, TREK-2, and TRAAK (TWIK-related arachidonic acid-activated K+ channel) mRNA, and the expression of these three proteins was confirmed by immunocytochemistry in mSCG neurons. IRIL was enhanced by zinc, inhibited by barium and fluoxetine, but unaffected by quinine and ruthenium red, strongly suggesting that it was carried through TREK-1/2 channels. Consistently, a channel with properties identical with the heterologously expressed TREK-2 was recorded in most (75%) cell-attached patches. These results provide the first evidence for the expression of K2P channels in the mammalian autonomic nervous system, and they extend the impact of these channels to the entire nervous system.

Development and characterization of 248 novel microsatellite markers in turbot (Scophthalmus maximus)

The turbot is a flatfish species of great relevance to marine aquaculture in Europe. Only a limited number of microsatellites have been isolated to date in this species. To increase the number of potentially useful mapping markers, we screened simple sequence repeat (SSR)--enriched genomic libraries obtained from several di-, tri-, and tetranucleotide tandem repeat motifs. A total of 248 new polymorphic microsatellites were successfully optimized. The efficiency of the protocol applied (6.4%) was higher than that in other studies of fish that used the same method. Dinucleotide and perfect microsatellites were predominant in this species; the (AC)n motif was the most frequent class of repeat. Polymorphism and structural properties at these loci, together with 30 variable loci previously reported in turbot, were evaluated in 6 wild individuals. The number of alleles per locus ranged from 2 to 10, with an average of 4.046. The microsatellite markers characterized in this study will contribute to the development of the turbot genetic map, which can be used for quantitative trait locus (QTL) identification, marker-assisted selection programs, and other applications to improve its culture.

Microsatellites of Mytilus chilensis: A Genomic Print of Its Taxonomic Status within Mytilus sp.

ABSTRACT The taxonomic status of the Chilean blue mussel Mytilus chilensis has been controversial for decades because of its phenotypic and genetic proximity to other species of the genus Mytilus from both hemispheres. This study reports the development of nine polymorphic microsatellite markers from the M. chilensis genome. The number of alleles per locus ranged between four and nine, and the observed heterozygosity ranged from 0.182–0.750 in a wild sample of 24 individuals from Caicaén (Chiloé Region, Chile). Lack of efficient amplification of many of these microsatellite loci in other Mytilus species suggests that M. chilensis is a valid, distinct species within the genus. These new markers would be useful in fine-scale population analyses of M. chilensis as well in the aquaculture management of this marine resource.

Expression of K2P Channels in Sensory and Motor Neurons of the Autonomic Nervous System

Several types of neurons within the central and peripheral somatic nervous system express two-pore-domain potassium (K2P) channels, providing them with resting potassium conductances. We demonstrate that these channels are also expressed in the autonomic nervous system where they might be important modulators of neuronal excitability. We observed strong mRNA expression of members of the TRESK and TREK subfamilies in both the mouse superior cervical ganglion (mSCG) and the mouse nodose ganglion (mNG). Motor mSCG neurons strongly expressed mRNA transcripts for TRESK and TREK-2 subunits, whereas TASK-1 and TASK-2 subunits were only moderately expressed, with only few or very few transcripts for TREK-1 and TRAAK (TRESK ≈ TREK-2 > TASK-2 ≈ TASK-1 > TREK-1 > TRAAK). Similarly, the TRESK and TREK-1 subunits were the most strongly expressed in sensorial mNG neurons, while TASK-1 and TASK-2 mRNAs were moderately expressed, and fewer TREK-2 and TRAAK transcripts were detected (TRESK ≈ TREK-1 > TASK-1 ≈ TASK-2 > TREK-2 > TRAAK). Moreover, cell-attached single-channel recordings showed a major contribution of TRESK and TREK-1 channels in mNG. As the level of TRESK mRNA expression was not statistically different between the ganglia analysed, the distinct expression of TREK-1 and TREK-2 subunits was the main difference observed between these structures. Our results strongly suggest that TRESK and TREK channels are important modulators of the sensorial and motor information flowing through the autonomic nervous system, probably exerting a strong influence on vagal reflexes.

Presence of two mitochondrial genomes in the mytilid Perumytilus purpuratus: Phylogenetic evidence for doubly uniparental inheritance

This study presents evidence, using sequences of ribosomal 16S and COI mtDNA, for the presence of two mitochondrial genomes in Perumytilus purpuratus. This may be considered evidence of doubly uniparental mtDNA inheritance. The presence of the two types of mitochondrial genomes differentiates females from males. The F genome was found in the somatic and gonadal tissues of females and in the somatic tissues of males; the M genome was found in the gonads and mantle of males only. For the mitochondrial 16S region, ten haplotypes were found for the F genome (nucleotide diversity 0.004), and 7 haplotypes for the M genome (nucleotide diversity 0.001), with a distance Dxy of 0.125 and divergence Kxy of 60.33%. For the COI gene 17 haplotypes were found for the F genome (nucleotide diversity 0.009), and 10 haplotypes for the M genome (nucleotide diversity 0.010), with a genetic distance Dxy of 0.184 and divergence Kxy of 99.97%. Our results report the presence of two well-differentiated, sex-specific types of mitochondrial genome (one present in the male gonad, the other in the female gonad), implying the presence of DUI in P. purpuratus. These results indicate that care must be taken in phylogenetic comparisons using mtDNA sequences of P. purpuratus without considering the sex of the individuals.

Identification of South Atlantic Hakes (Merluccius australis and Merluccius hubbsi)in Processed Foods by PCR-RFLPs of Cytochrome bGene

Abstract The application of molecular genetics to the identification of fishes is an appropriate technology for quality control and commercial tracking. We have applied a DNA-based methodology to identify the exact species of Argentinean hake included in commercial products. The first test consisted in the PCR amplification of a 122 bp fragment from the cytochrome bgene to assess the presence of DNA from hakes. This test being positive, it was followed by a second test consisting of the amplification and digestion of a PCR fragment of 464 bp from the cytochrome bgene, to determine the identity of the commercialized species. Since a single restriction enzyme provided full diagnosis of the southern Atlantic hake species present in the samples, this methodology would be of great help in traceability across the food-chain that comprises (1) the authentication of Argentinean hake products, (2) the control of fraud to consumers, (3) the industrial advice regarding the labeling of commercial products, and (4) the fishery forensics of Argentinean hakes.

Molecular Cytogenetic Analysis of the European Hake Merluccius merluccius (Merlucciidae, Gadiformes): U1 and U2 snRNA Gene Clusters Map to the Same Location

The European hake (Merluccius merluccius) is a highly valuable and intensely fished species in which a long-term alive stock has been established in captivity for aquaculture purposes. Due to their huge economic importance, genetic studies on hakes were mostly focused on phylogenetic and phylogeographic aspects; however chromosome numbers are still not described for any of the fifteen species in the genus Merluccius. In this work we report a chromosome number of 2n = 42 and a karyotype composed of three meta/submetacentric and 18 subtelo/telocentric chromosome pairs. Telomeric sequences appear exclusively at both ends of every single chromosome. Concerning rRNA genes, this species show a single 45S rDNA cluster at an intercalary location on the long arm of subtelocentric chromosome pair 12; the single 5S rDNA cluster is also intercalary to the long arm of chromosome pair 4. While U2 snRNA gene clusters map to a single subcentromeric position on chromosome pair 13, U1 snRNA gene clusters seem to appear on almost all chromosome pairs, but showing bigger clusters on pairs 5, 13, 16, 17 and 19. The brightest signals on pair 13 are coincident with the single U2 snRNA gene cluster signals. Therefore, the use of these probes allows the unequivocal identification of at least 7 of the chromosome pairs that compose the karyotype of Merluccius merluccius thus opening the way to integrate molecular genetics and cytological data on the study of the genome of this important species.